Bioanalytical assays such as nucleic acid hybridization assays and immunoassays are extremely important in a variety of fields, for example diagnostic medicine, forensics, genetics, and drug and agricultural testing. The use of enzymes as labelling groups in such assays is prevalent due to the intrinsic amplification provided by the activity of the enzyme in producing a large number of detectable product molecules per molecule of enzyme. In addition, assays of biological samples for specific enzymatic activity are widely used in diagnostic medicine as well as in biological and medical research fields. It is always desirable to improve the sensitivity of such assays. One way to do so is to improve the detectability of the product molecule, and hence the detectability of the enzyme itself, whether the enzyme is the analyte or is used as a labelling group.
Currently such assays are typically performed using colorimetric, chemiluminescence or fluorescence detection, in which a colorless or nonluminescent substrate is converted into a colored, chemiluminescent or fluorescent product, respectively, by the action of the enzyme. The product of the enzymatic reaction can be measured in solution, or alternately can be measured after deposition onto a solid phase such as a membrane or polystyrene support. Colorimetric assays are generally the least sensitive due to the poor sensitivity intrinsic to measurement of light absorption, which results in poor detectability of the product. The measurement of light emission is intrinsically a more sensitive technique and the theoretical detectability of chemiluminescent or fluorescent products is better. Chemiluminescent detection is in fact quite sensitive but is limited to the use or detection of a few specific enzymes due to the limited number of reactions currently known which form chemiluminescent products. Fluorescence detection can be used with a much wider variety of enzymes, but conventional fluorescence detection suffers from difficulty in discriminating specific fluorescence signal from nonspecific background signals, which restrict the practical detection limit of an assay. A number of such methods and their use in immunoassays and nucleic acid hybridization assays are reviewed in the following references: GJR Barnard & WP Collins, Med. Lab. Sci. (1987) 44 249-66; A Johannson, DH Ellis, DL Bates, AM Plumb, CJ Stanley, J. Immunol. Meth. (1986) 87 7-11; EP Diamandis, Clin. Biochem. (1988) 87 139-50; M Oellerich, Meth. Enzym. Anal. Vol. I 1983 233-60; CG Guilbault, Pure Appl. Chem. (1985) 57 495-514; and JA Matthews & LJ Kricka Anal. Biochem. (1988) 169 1-25.
Time-resolved luminescence detection of lanthanide chelates has been utilized in order to decrease nonspecific background signals. Background signals from other light emission or scattering processes (resulting from scattering of the excitation light) can be reduced by taking advantage of the unusual luminescence characteristics of lanthanide chelates, particularly of europium and terbium. Such chelates can exhibit strongly red-shifted, narrow-band, long-lived emission after excitation of the chelate at substantially shorter wavelengths. Typically, the chelate possesses a strong ultraviolet absorption band due to a chromophore which is part of the chelating molecule and located close to the lanthanide in the chelate. Subsequent to light absorption by the chromophore, if the energy levels of the chromophore and lanthanide are suitably matched, the excitation energy can be transferred from the excited chromophore to the lanthanide. This is followed by luminescence emission characteristic of the lanthanide. The use of pulsed excitation and time-gated detection combined with narrow-band emission filters allows for specific detection of the luminescence from the lanthanide chelate only, rejecting emission from other species present in the sample which are typically shorter-lived or have shorter wavelength emission. In currently existing assay formats using this principle, a lanthanide chelating agent is utilized as the labelling group. In some cases the chelating agent itself forms a luminescent lanthanide chelate which is measured directly and is usually bound by some method to a solid support. Such methods are outlined in the following patents: U.S. Pat. No. 4,374,120 (Soini & Hemmila), U.S. Pat. No. 4,637,988 (Hinshaw et al.), EP 0 203 047 (Kankare & Takalo), U.S. Pat. No. 4,259,313 (Frank & Sundberg), U.S. Pat. No. 4,058,732 (Wieder), and U.S. Pat. No. 4,772,563 (Evangelista & Pollak). In other cases the lanthanide must be released into solution to be measured in the presence of luminescence enhancing reagents, usually consisting of a combination of a detergent and a weakly complexing energy transfer reagent as illustrated in patents U.S. Pat. No. 4,565,790 (Hemmila & Dakubu) and WO 89/04375 (Musso et al.). Luminescence detection of certain lanthanide chelates has also been disclosed in EP 0 195 413 (Hale & Solas).